XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis

Breast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women. Despite improvement in diagnosis, the survival rate of this disease has still not improved.

Hussain et al BMC Cancer (2017) 17:640 DOI 10.1186/s12885-017-3627-4 RESEARCH ARTICLE Open Access XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis Azhar R Hussain1†, Abdul Khalid Siraj1†, Maqbool Ahmed1†, Rong Bu1, Poyil Pratheeshkumar1, Alanood M Alrashed2, Zeeshan Qadri1, Dahish Ajarim3, Fouad Al-Dayel4, Shaham Beg1 and Khawla S Al-Kuraya1,2* Abstract Background: Breast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women Despite improvement in diagnosis, the survival rate of this disease has still not improved X-linked Inhibitor of Apoptosis (XIAP) has been shown to be over-expressed in various cancers leading to poor overall survival However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored Methods: We examined the expression of XIAP in more than 1000 Middle Eastern breast cancer cases by immunohistochemistry Apoptosis was measured by flow cytometry Protein expression was determined by western blotting Finally, in vivo studies were performed on nude mice following xenografting and treatment with inhibitors Results: XIAP was found to be over-expressed in 29.5% of cases and directly associated with clinical parameters such as tumor size, extra nodal extension, triple negative breast cancer and poorly differentiated breast cancer subtype In addition, XIAP over-expression was also significantly associated with PI3-kinase pathway protein; p-AKT, proliferative marker; Ki-67 and anti-apoptotic marker; PARP XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis Next, we investigated inhibition of XIAP using a specific inhibitor; embelin and found that embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells Finally, breast cancer cells treated with combination of embelin and PI3-kinase inhibitor; LY294002 synergistically induced apoptosis and caused tumor growth regression in vivo Conclusion: These data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells Keywords: Breast cancer, XIAP, Embelin, P-AKT, Apoptosis * Correspondence: kkuraya@kfshrc.edu.sa † Equal contributors Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Cancer, MBC#98-16, P.O Box 3354, Riyadh 11211, Saudi Arabia AlFaisal University, Riyadh, Saudi Arabia Full list of author information is available at the end of the article © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Hussain et al BMC Cancer (2017) 17:640 Background Breast cancer is the most common cancer in females and despite improvement in treatment modality, the overall survival rate of breast cancer remains low [1] Even though, incidence of breast cancer increases with age [2], it has been seen that there is trend towards an increase in incidence of breast cancer in younger women in western countries as well as Middle Eastern region [3–5] In Saudi Arabia, breast cancer is the most common cancer in females as well as remains the major cause of morbidity and mortality within the female population [6] One reason behind this increase in morbidity and mortality in breast cancer could be the strong-association with many aggressive molecular markers that tend to cause increased proliferation of cancer cells and impart resistance to conventional chemotherapy [7, 8] These aggressive markers include dysregulated proteins of the survival pathways [8] and proliferative markers [9] that tend to make the tumor resistant to conventional chemotherapy, grow rapidly and spread to surrounding tissues and distant organs For these reasons, there is an urgent need for identifying molecular targets that are either overexpressed or constitutively activated in breast cancer that can be therapeutically targeted Inhibitor of Apoptosis Proteins (IAPs) family is slowly emerging as viable therapeutic targets for the treatment of cancer because of their ability to be selectively overexpressed in various cancers as compared to their normal counterparts [10, 11] Of the many members of the family, X-linked Inhibitor of Apoptosis Protein (XIAP) has been found to be the most promising target because XIAP is found to be over-expressed in a variety of cancers [12–15] In addition, XIAP over-expression also leads to poor prognosis in many cancers including breast and thyroid cancer [14, 16] Structurally, XIAP contains three tandem 80 amino acid repeats known as baculovirus IAP repeats (BIR) and a zinc ring domain that contains the E3 ligase ubiquitin activity thereby making XIAP susceptible to ubiquitination [17, 18] The main role of XIAP is to disrupt and inhibit apoptosis by acting at caspase-3 and -7 via the second BIR domain and caspase-9 via the third BIR domain [19–21] Because of the anti-apoptotic effect as well as its over-expressing potential in cancer cells as compared to its normal counterparts, XIAP is emerging as a potential therapeutic target for the management of cancer There are several XIAP inhibitors have been reported and some are in clinical trial [22–24] Embelin is the only natural, cell-permeable, non-peptide small molecule XIAP inhibitor reported so far [25, 26] It selectively inhibits the growth of cancer cells and induces apoptosis, with nontoxic or low-toxic to normal cells [27] Embelin binds to the BIR3 domain of XIAP and block the interaction of XIAP with caspases to promote apoptosis [28] Page of 13 Survival of cancer cells is necessary for their propagation, invasion and migration leading to their disruptive behavior and damage to the normal working environment of the human body This is usually achieved by not only over-expression of anti-apoptotic proteins but also by causing dysregulation of various signaling transduction pathways [29] One pathway that is found to be dysregulated in many cancers is the PI3-kinase/AKT pathway whereby constitutive activation of survival protein, AKT promotes survival via inhibiting the apoptotic pathway, increased glucose metabolism and promote proliferation [30–32] The PI3-kinase/AKT pathway has therefore been the target of many new experimental therapeutic agents because of its pro-survival and antiapoptotic role in many cancers However, the success of managing these cancers with single agents has been limited [33, 34] On the other hand, PI3-kinase inhibitors have been more successful in combination with either other inhibitors or chemotherapeutic agents via sensitizing cancers cells to undergo apoptosis [35, 36] In this study, we have investigated expression of XIAP in a large cohort of more than 1000 clinical breast cancer samples in tissue microarray (TMA) format by immunohistochemistry and determined the association of XIAP over-expression with various clinical parameters and molecular markers This is followed by in vitro and in vivo targeting of XIAP in breast cancer cells using specific XIAP inhibitor, embelin, either alone or in combination with PI3-kinase/AKT inhibitor, LY294002 to assess cell viability, apoptosis and xenograft tumor regression Methods Patient selection and tissue microarray (TMA) construction Samples from 1009 breast cancer (BC) patients diagnosed between 1990 and 2011 were identified and selected from the tissue bio-repository of King Faisal Specialist Hospital and Research Centre (KFSHRC) Detailed clinico-pathological data, including survival data, were noted from case records Follow-up was calculated from the date of resection of the primary tumor, and all surviving cases were censored for survival analysis on 31 December 2011 Three pathologists reviewed all tumors for grade and histological subtype All BC samples were analyzed in a tissue microarray (TMA) format TMA construction was performed from formalin-fixed, paraffin-embedded BC specimens and slides were processed and stained manually as described previously [37] Briefly, tissue cylinders with a diameter of 0.6 mm were punched from representative tumor areas of a ‘donor’ tissue block using a semi-automatic robotic precision instrument and brought into different recipient paraffin blocks each containing between 133 and 374 Hussain et al BMC Cancer (2017) 17:640 individual samples A block containing normal and tumor tissue from multiple organ sites was used as control Institutional Review Board (IRB) and Research Ethics Committee (REC) of KFSHRC approved the study under the Project RAC#2040 004 on BC archival clinical samples along with a waiver of consent and Project RAC#2110 025 for animal studies Immunohistochemistry Primary antibody clones and their dilutions used for IHC are given in Additional file 1: Table S1 XIAP, PARP, Ki-67 and p-AKT expression were analyzed by immunohistochemistry on a TMA as described before [12] X-tile plots were constructed for assessment of biomarker and optimization of cut off points based on outcome as has been described earlier [38] Based on XIAP expression, BCs were grouped into groups based on X-tile plots: one with complete absence or reduced staining (H score = 0–85) and the other group showing over expression (H score > 85) Statistical analysis Contingency table analysis and chi-square tests were used to study the relationship between clinicopathological variables and XIAP Overall survival curves were generated using the Kaplan-Meier method, with significance evaluated using the Mantel-Cox log-rank test The limit of significance for all analyses was defined as a p-value of 0.05; two-sided tests were used in all calculations The JMP 9.0 (SAS Institute, Inc., Cary, NC) software package was used for data analyses Cell culture Breast cancer (BC) cell lines, CAL-120 (ACC 459) was obtained from DSMZ (Braunschweig, Germany) EVSAT (CSC-C0468) was purchased from Creative Bioarray (NY, USA) MCF7 (ATCC® HTB-22™) and MDA-MB231 (ATCC® HTB-26™) were obtained from ATCC (Manassas, VA) All the cell lines were cultured in RPMI 1640 media supplemented with 10% Fetal Bovine Serum (FBS), Pen-Strep and Glutamine as described previously [30] All experiments were performed using 5% FBS in RPMI 1640 media All the cell lines were authenticated in house using short tandem repeats PCR Reagents and antibodies Embelin was purchased from Tocris Bioscience (Ellisville, MO) MTT was purchased from Sigma (St Louis MO, MA) LY294002 and zVAD-fmk was purchased from Calbiochem (San Diego, CA, USA) XIAP antibody was purchased from BD Transduction lab (San Jose, CA, USA) Antibodies against caspase-9, caspase-3, PARP, p-AKT, p-Bad, Bcl-2, Bcl-Xl, Beta-actin, Survivin and Bid were purchased from Cell Signaling Technologies Page of 13 (Beverly, MA, USA) Cytochrome c and GAPDH antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA) cIAP-1 antibody was purchased from R&D (USA) Annexin V/PI staining kit was purchased from Molecular Probes (Eugene OR, USA) Cell lysis and immunoblotting Following treatment with inhibitors or siRNA, BC cells were lysed and proteins were isolated as previously described [39] Following protein isolation, equal amount of protein were separated by SDS-Page and immunoblotted with different antibodies The blots were developed using enhanced chemiluminescence (ECL, Amersham, Illinois, USA) system 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) assays BC cells were plated at a density of 104 cells for 24 h in 96 well plates and were treated with different doses of embelin or LY294002 for 24 h at a final volume of 200 μl MTT assays were performed to determine cell viability using a plate reader as previously described [40] Results depicted are from three independent experiments *p < 0.05 and **p < 0.005 Cell cycle analysis and apoptosis assay For cell cycle analysis and annexin V/PI staining for apoptosis, following treatment with 25 and 50 μM embelin, cells were harvested and washed with 1× PBS and re-suspended in either 500 μl hypotonic staining buffer for cell cycle analysis or annexin V/PI for apoptosis assay Following incubation, cells were analyzed by flow cytometry as shown before [41] Assay for cytochrome c release Following treatment with embelin for 24 h, mitochondrial free cytosolic extracts and cytosolic free mitochondrial extracts were isolated as described previously [30] Equal amount of protein (20 μg) were separated with SDS-Page and immunoblotted with antibodies against anti-cytochrome c, GAPDH and Cox IV antibodies Measurement of mitochondrial potential Following treatment with Embelin, cells were stained with JC1 dye and incubated at 37 °C for 30 in the dark After incubation, cells were washed, re-suspended in PBS and analyzed by flow cytometry as described early [42] Gene silencing using siRNA XIAP siRNA (Cat no 6550 and 6446) were purchased from Cell Signaling, AKT siRNA (Cat no SI02757244 and SI02758406) as well as Scrambled control siRNA (Cat no 1027281) were purchased from Qiagen (Valencia, CA, USA) siRNA were transfected into breast cancer cell lines Hussain et al BMC Cancer (2017) 17:640 as described previously [12] Following 48 h transfection, proteins were isolated and expression was determined by Western Blot analysis Animals and xenograft study Female nude mice were chosen for these experiments and mice were injected with MDA-MB-231 cells (10 million per animal) Following one week of injection, the animals were randomly assigned into three groups The first groups were not treated and only vehicle (DMSO) was injected while the other two groups were treated with 10 and 20 mg/kg embelin, injected intra-peritoneally, twice weekly for weeks respectively In the second set of experiments, the female mice were divided into four groups, the first group received DMSO alone, while the second and third received embelin (10 mg/kg) and LY294002 (10 mg/kg) The fourth group received a combination of embelin and LY294002, injected simultaneously During the study, the weight and tumor volume of each mouse was monitored weekly After weeks of treatment, mice were sacrificed and individual tumors were weighed, and then snap frozen in liquid nitrogen for storage Results Determination of XIAP expression by IHC and correlation with clinical data and molecular markers To identify the role of XIAP in the pathogenesis of breast cancer, we analyzed expression of XIAP by IHC on a TMA format on a large cohort of BC samples collected at KFSHRC from 1990 to 2011 Our data showed that 29.5% (284/964) BC samples had over-expression of XIAP (Table 1) Clinically, XIAP over-expression was significantly associated with tumor size (p = 0.0044), extra-nodal extension (p = 0.0041), poorly differentiated tumor (p < 0.0001), triple negative breast cancer (0.0019) and infiltrative ductal carcinoma subtype (p = 0.002) At the molecular level, XIAP over-expression significantly associated with proliferative marker; Ki67 (p < 0.0001), PARP (p < 0.0001) and p-AKT (p < 0.0001) (Table and Fig 1a) Finally, XIAP over-expression led to a poor overall survival of 71.8% as compared to 82.8% (p = 0.0005) (Fig 1b) and was found to be an independent poor prognostic marker in multivariate analysis (Additional file 2: Table S2) Down-regulation of XIAP using embelin inhibited cell viability and induced apoptosis in BC cells Our clinical data showed that XIAP over-expression was associated with a significant year poor survival of 71.8% (p = 0.005) (Table 1) Therefore, we wanted to investigate whether XIAP could be targeted using a specific XIAP inhibitor, embelin [28] to inhibit cell growth and induce apoptosis in BC cells Therefore, we treated Page of 13 four BC cell lines; CAL-120, EVSAT, MCF-7 and MDAMB-231 with increasing doses of Embelin for 24 h to assess cell viability using MTT assay As shown in Fig 2a, Embelin inhibited cell viability in all the four cell lines that expressed XIAP in a dose dependent manner Next, to determine whether embelin induced cell inhibition was due to apoptosis, we treated BC cells with increasing doses of embelin for 24 h and analyzed the cells for apoptosis after dual staining with annexin V/PI by flow cytometry As shown in Fig 2b, all the four BC cell lines underwent apoptosis at increasing doses however the IC50 of all four cell lines ranged between 25 and 50 μM concentration of embelin and therefore, the rest of the experiments were performed at 25 and 50 μM only Once, it was ascertained that the BC cells were undergoing apoptosis following embelin treatment, we wanted to determine whether embelin treatment of BC cells downregulated expression of XIAP and induced caspase dependent apoptosis Therefore we chose two cell lines; EVSAT and MDA-MB-231 and treated them with 25 and 50 μM embelin for 24 h Following treatment, proteins were isolated and probed with antibodies against XIAP, caspases-9 and -3, PARP and GAPDH Our data showed that embelin treatment caused down-regulation of XIAP expression and cleavage of caspases-9 and -3 in both the cells as demonstrated by decreased intensity of pro-bands In addition, embelin treatment also induced cleavage of PARP, a protein that needs to be cleaved for efficient apoptosis to occur [43, 44] (Fig 2c) To confirm these findings, we also transfected EVSAT and MDAMB-231 with either non-specific scrambled siRNA or siRNA targeted against XIAP and assessed the protein expression following transfection by immunoblotting As shown in Fig 2d, we found similar results with downregulation of XIAP thereby confirming the role of embelin in inducing caspase-dependent apoptosis in BC cells XIAP down-regulation was also confirmed using another XIAP siRNA (Data not shown) Embelin treatment also transcriptionally down-regulated expression of XIAP in EVSAT cells as detected by real-time RT-PCR (Fig 2e) Furthermore, we also pre-treated MDA-MB-231 cells with a universal caspase-inhibitor, zVAD-fmk for three hours followed by treatment with 50 μM embelin for 24 h As shown in Fig 2f, zVAD-fmk pre-treatment restored expression of caspases-9, −3 and inhibited PARP breakdown in BC cells This data confirmed that embelininduced apoptosis is caspase dependent Embelin treatment activated mitochondrial apoptotic pathway via in-activation of AKT in BC cells Our clinical data on the cohort of BC samples showed a significant association between XIAP expression and activated AKT In addition, we and others have also shown that XIAP expression and activated AKT are closely Hussain et al BMC Cancer (2017) 17:640 Page of 13 Table Correlation of XIAP with clinico-pathological parameters in Breast Cancer Total N Total Number of Cases % 964 XIAP Over-expression XIAP Low-expression N % N % 284 29.5 680 70.5 P value Age Groups < 50 306 31.7 87 28.4 219 71.6 > 50 658 68.3 197 29.9 461 70.1 0.6320 Tumor sizea ≤ cm 208 22.1 46 22.1 162 77.9 > cm 731 77.9 236 32.1 498 67.9 Negative 300 33.3 81 27.0 219 73.0 Positive 602 66.7 179 29.7 423 70.3 M0 776 89.8 225 29.0 551 71.0 M1 88 10.2 32 36.4 56 63.6 I 76 9.1 19 25.0 57 75.0 II 366 43.7 107 29.2 259 70.8 III 307 36.7 91 29.6 216 70.4 IV 88 10.5 32 36.4 56 63.6 Present 262 33.2 92 35.1 170 64.9 Absent 527 66.8 133 25.2 394 74.8 Present 350 41.0 110 31.4 240 68.6 Absent 504 59.0 135 26.8 369 73.2 Well differentiated 72 7.6 10 13.9 62 86.1 Moderately differentiated 489 51.3 123 25.1 366 74.9 Poorly differentiated 393 41.2 150 38.2 243 61.8 0.0044 Lymph Nodes involvementa 0.3914 Metastasisa 0.1587 Tumor Stagea 0.4453 Extra Nodal Ext.a 0.0041 LVIa 0.1411 Histological Grade a